EP1627921A1 - Method for detecting nucleic acid amplification in real-time comprising positioning a reaction vial relative to a detection unit - Google Patents

Method for detecting nucleic acid amplification in real-time comprising positioning a reaction vial relative to a detection unit Download PDF

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Publication number
EP1627921A1
EP1627921A1 EP04019542A EP04019542A EP1627921A1 EP 1627921 A1 EP1627921 A1 EP 1627921A1 EP 04019542 A EP04019542 A EP 04019542A EP 04019542 A EP04019542 A EP 04019542A EP 1627921 A1 EP1627921 A1 EP 1627921A1
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EP
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Prior art keywords
reaction
fluorescent dye
fluorescence
amplification
reaction mixture
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EP04019542A
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German (de)
French (fr)
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EP1627921B1 (en
Inventor
Christian Dr. Weilke
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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Priority to AT04019542T priority Critical patent/ATE406462T1/en
Priority to DE502004007951T priority patent/DE502004007951D1/en
Priority to EP04019542A priority patent/EP1627921B1/en
Priority to US11/203,845 priority patent/US20060040303A1/en
Publication of EP1627921A1 publication Critical patent/EP1627921A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/11—Filling or emptying of cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/13—Moving of cuvettes or solid samples to or from the investigating station
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0325—Cells for testing reactions, e.g. containing reagents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/0332—Cuvette constructions with temperature control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/08—Optical fibres; light guides

Definitions

  • the present invention relates to a system for improved optical measurement of analytical reactions, in particular the measurement of real-time PCR amplification reactions by means of fluorescence measurement.
  • the LighCycler device is a Real Time Thermocycler instrument that uses a stream of air to heat or cool capillary reaction vessels.
  • the excitation of fluorescence-labeled hybridization probes or intercalating dyes is carried out with the aid of a light-emitting diode (LED), the detection of fluorescence by means of a fluorimeter containing multiple photo-hybrids for the detection of different wavelengths (WO 97/46712).
  • LED light-emitting diode
  • both the excitation and the measurement of the emitted light take place by means of a beam path parallel to the longitudinal axis of the capillary (FIG. 1).
  • the excitation light is irradiated through the lower end of the capillary into the reaction vessel.
  • a large proportion of the emitted light is reflected at the Kapillarenwandung and passes in the opposite direction again through the lower capillary end for detection on the photo-hybrid.
  • the capillaries are in a rotatable carousel as a reaction vessel holder, which is suitable for receiving 32 capillaries.
  • each The capillary can be positioned in a tangential direction using a first stepper motor rotating the carousel.
  • a second stepper motor can position the fluorimeter in a specific position relative to the respective capillary to be measured in the radial direction.
  • LightCycler instruments of all previous instrument versions determine the optimal position for the fluorescence measurement ("Seek") for each individual capillary at the beginning of an experiment.
  • the reaction mix filled capillary in the capillary fixture i.
  • the procedure continues in small steps and the point of maximum fluorescence is determined.
  • the fluorimeter is displaced at right angles with a second stepper motor and the position of maximum fluorescence is also determined. This x, y position is stored for each capillary and restarted at each subsequent measurement.
  • the capillaries used as reaction vessels are so thin that a high signal intensity is measured only in a very limited range. Because of variances in the capillary shape and the carousels, such a seek must be performed first for each experiment (LightCycler Operator's Manual, Version 3.5, Oct. 2000).
  • the reaction mixture must have a sufficiently high background fluorescence even before the PCR, otherwise no maximum of the fluorescence signal can be determined.
  • the basic fluorescence can be so low that capillaries can not be positioned in the LightCycler instrument.
  • the relative position of the individual reaction vessel to the detection unit can be changed by changing the position of the reaction vessel holder.
  • the relative position can also be changed by changing the position of the detection unit.
  • both positions can be changed for exact positioning.
  • ABI uses the reference dye (ROX) to normalize the signal levels of various tubes to compensate for variances in the optical measurement unit, microtiter plates, and composition and fill levels of the reagent mixes
  • Such systems preferably contain capillaries as reaction vessels.
  • the present invention furthermore relates to a reaction mixture comprising thermostable polymerase, deoxynucleotides, buffers and additionally a fluorescent dye component independent of the amplification reaction and having an emission wavelength of at least 600 nm.
  • such mixtures preferably contain at least two amplification primers and at least one fluorescence-labeled hybridization probe, for example a TaqMan probe.
  • kits comprising thermostable polymerase, deoxynucleotides and buffers and additionally to a fluorescence dye component independent of the amplification reaction having an emission wavelength of at least 600 nm.
  • kits may additionally contain specific hybridization probes such as TaqMan probes or also amplification primers.
  • fluorescent dye component initially includes any type of free, unbound fluorescent dye, with long-wave fluorescent dyes having an emission maximum of more than 600 nm have proven to be particularly advantageous because the measurement is not affected in the shorter wavelength detection channels.
  • fluorescent dye component also includes fluorescent dyes which are coupled to another chemical component, if such molecules do not interfere with the Amplification reaction interfere and not be incorporated during the reaction in the amplicon.
  • the one or more means for positioning multiple reaction vessels in a reaction vessel holder relative to the detection unit may according to the invention be any type of finely adjustable drive.
  • these are one or more stepper motors, which can change in very small steps either the position of the reaction vessel holder or the position of the detection unit or both.
  • the detection unit may be any type of detectors capable of detecting fluorescence signals. These include, for example, fluorimeter with one or more photo-hybrid diodes or CCD cameras.
  • the LightCycler instrument described above can be used for various detection formats:
  • the respective amplification product is detected in this case by a DNA binding dye, which emits a corresponding fluorescence signal upon interaction with double-stranded nucleic acid after excitation with light of a suitable wavelength.
  • a DNA binding dye which emits a corresponding fluorescence signal upon interaction with double-stranded nucleic acid after excitation with light of a suitable wavelength.
  • a DNA binding dye particularly suitable for this application are the dyes SybrGreen and SybrGold (Molecular Probes) proved.
  • SybrGreen and SybrGold Molecular Probes
  • two single-stranded hybridization probes are used simultaneously which are complementary to adjacent sites of the same strand of amplified target nucleic acid. Both probes are labeled with different fluorophores, which differ in their absorbance and emission wavelengths. When the shorter-wavelength fluorophore is excited, almost exclusively the fluorescence of this fluorophore can be measured without target nucleic acid. If both probes hybridize to amplified target nucleic acid, the two fluorophores approach so far that the excited fluorophore can transfer the absorbed energy to the second fluorophore according to the principle of fluorescence resonance energy transfer and its emission in the long-wave detection channel becomes measurable (WO 97/46707).
  • donor component for example, fluorescein is excited with light of a suitable wavelength.
  • acceptor component such as certain rhodamine derivatives
  • resonance energy transfer then takes place to the acceptor component, so that the acceptor fluorophore emits light of a higher emission wavelength.
  • the emission of the FRET donor is sufficient to perform a positioning process.
  • a single-stranded hybridization probe is labeled with 2 components.
  • the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer, so that the radiation emission of the first component is almost completely suppressed.
  • the hybridization probe binds to the target DNA and is cleaved upon primer elongation by the 5'-3 'exonuclease activity of the Taq polymerase. This will be the excited fluorescent component and the quencher are spatially separated so that a fluorescence emission of the first component at the corresponding wavelength can be measured (US 5,210,015, US 5,487,972, US 5,804,375, US 6,214,979).
  • a passive fluorescence dye (“Seek Dye”) is added to the reaction mixture, which does not affect the PCR and optical detection is not disturbs.
  • the reaction mixture contains a fluorescent dye component which is in free form during and after the entire amplification reaction. This means that the dye component is not covalently incorporated into the amplification products in the form of labeled primers or labeled dNTPs during amplification and moreover also has no binding affinity for the double-stranded DNA amplification product, as is the case with SybrGreen, for example.
  • the emission maximum of this passive Seek Dye is preferably in the long-wavelength range of more than 600 nm and particularly preferably in the longest-wave detection channel of the six-channel LightCycler 2.0 instrument.
  • the detection in the shorter wave channels 530-640 nm, in LightCycler TaqMan assays due to the commercially available and suitable dyes almost exclusively used will not be affected.
  • In the channels 705 and 670 nm only a constant increase of the background signal is visible, which usually does not interfere with the measurement of signal level changes.
  • the Seek Dye has a sufficiently high stability against temperature and light exposure, so that the properties do not change during the PCR.
  • the use of JA286 EP 0 747 447, example 1 has proven to be particularly preferred.
  • the system according to the invention preferably contains capillaries as reaction vessels.
  • the reaction mixture already contains at least two amplification primers and at least one hybridization probe, for example a TaqMan hybridization probe.
  • the system preferably contains as fluorescent dye component a fluorescent dye with an emission maximum of more than 600 nm, for example the fluorescent dye JA 286 (EP 0 747 447, example 1).
  • the positioning process (Seek procedure) in the Light Cycler can be as follows for a single capillary:
  • the closed chamber is heated to 30 ° C or another temperature set in Run.
  • a home run is performed in which the photoelectric sensors and rotor are approached as the reference position. Thereafter, the position of the maximum fluorescence signal is determined for each sample. The measurement takes place in all existing detection channels, the channel with the highest signal is evaluated
  • the setpoint position for capillary 1 is stored as CarOffset and RadOffset. Around this position, a window is stretched, in which the Seek for capillary 1 takes place. The measurement takes place along 4 crossing tracks ('#'), therefore this process is called cross-seek. For each following capillary, the sample carousel is rotated further from the respectively determined position by 1/32 of the circumference and again the cross-seek is performed.
  • the carousel position of the maximum signal is determined and stored within the defined window around the approached position.
  • the photometer position of the maximum signal is determined and stored within the defined window.
  • the photometer position of the maximum signal is again determined and stored within the new window around the photometer position from phase 2.
  • this capillary Only if the maximum fluorescence exceeds a given minimum value, this capillary is declared as found. If the signal is too low, this capillary is considered not found. This avoids that positions without reaction mixture are nonsensitively positioned or incorrectly evaluated. If the capillary is found, the determined coordinates of rotor and photometer of phase-4 are stored and approached each time during the measurement of the capillary in the following RUN.
  • the present reaction mixture containing thermostable polymerase, deoxynucleotides comprises buffers, additionally containing a fluorescent dye component which is in free form during and after the entire amplification reaction.
  • a fluorescent dye component which is in free form during and after the entire amplification reaction.
  • this is a dye having an emission wavelength of more than 600 nm.
  • Particularly preferred is the fluorescent dye JA286.
  • Such mixtures can be prepared as master mixes for multiple reactions. After appropriate aliquoting such mixtures may be provided before the final use in an amplification reaction with at least 2 specific amplification primers and with at least one fluorescently labeled hybridization probe such as a TaqMan probe.
  • the present invention furthermore relates to kits comprising thermostable polymerase, deoxynucleotides and buffers, and additionally a fluorescent dye component having an emission wavelength of at least 600 nm, which is present in completely free form during and after the entire amplification reaction.
  • a fluorescent dye component having an emission wavelength of at least 600 nm, which is present in completely free form during and after the entire amplification reaction.
  • this is also a Dye with an emission wavelength of more than 600 nm.
  • the fluorescent dye JA286 is particularly preferred.
  • kits There are two types of kits:
  • Generic kits contain no additional components and can therefore be used for any type of real-time PCR, regardless of the detection format used and regardless of the target sequence to be amplified. The end user must add in this case still on an individual basis primers and hybridization probes according to his requirements.
  • parameter-specific kits usually contain at least two amplification primers and at least one fluorescence-labeled hybridization probe, preferably a TaqMan probe.
  • kits contain at least two amplification primers, at least one fluorescence-labeled hybridization probe and, according to the invention, a fluorescent dye component.
  • the amplified product was detected using the following TaqMan probe in real-time mode:

Abstract

A method for measuring nucleic acid amplification in real time which uses a real time Polymerase Chain Reaction (PCR) instrument with capillary tubes as reaction vessels, is new. A fluorescent dye is added to the reaction mixture and remains unchanged throughout the reaction. The capillaries are adjusted to the position where the fluorescence has a maximum value and fluorescence emission is carried out during and optionally after the reaction. Independent claims are also included for: (A) a system comprising a real time PCR instrument with several capillary tubes as reaction vessels, a reaction mixture comprising a thermostable polymerase, deoxynucleotide, buffer and a fluorescent dye which remains unchanged throughout the reaction; (B) a reaction mixture comprising a thermostable polymerase, deoxynucleotide, buffer and a fluorescent dye with an emission wavelength of at least 600nm which remains unchanged throughout the reaction; and (C) a kit comprising the reaction mixture.

Description

Technisches Gebiet:Technical area:

Die vorliegende Erfindung betrifft ein System zur verbesserten optischen Messung von analytischen Reaktionen, insbesondere die Messung von Echtzeit PCR Amplifikationsreaktionen mithilfe von Fluoreszenzmessung.The present invention relates to a system for improved optical measurement of analytical reactions, in particular the measurement of real-time PCR amplification reactions by means of fluorescence measurement.

Stand der Technik:State of the art:

Real Time PCR Verfahren gehören heutzutage zu den etablierten Standard-Methoden zur Analyse von Nukleinsäuren in vielen Molekularbiologischen Forschungslaboren und Diagnostischen Einrichtungen. Zur Durchführung derartiger Verfahren werden verschiedene instrumentelle Plattformen von diversen Herstellern auf dem Markt angeboten.Real-time PCR methods are nowadays one of the established standard methods for the analysis of nucleic acids in many molecular biology research laboratories and diagnostic institutions. To carry out such methods, various instrumental platforms are offered by various manufacturers on the market.

Ein seit mehreren Jahren etabliertes System ist das LightCycler Instrument (Roche Applied Science Cat.No. 03 531 414 201). Bei dem LighCycler Gerät handelt es sich um ein Real Time Thermocycler Instrument, bei dem kapillare Reaktionsgefäße mithilfe eines Luftstroms erhitzt bzw. abgekühlt werden. Die Anregung fluoreszenzmarkierter Hybridisierungssonden oder interkalierender Farbstoffe erfolgt mithilfe einer Licht emittierenden Diode (LED), die Detektion der Fluoreszenz mithilfe eines Fluorimeters, enthaltend multiple Photohybride zur Detektion verschiedener Wellenlängen (WO 97/46712). Dabei erfolgt sowohl die Anregung als auch die Messung des emittierten Lichts mithilfe eines Strahlengangs parallel zur Längsachse der Kapillare (Fig. 1). Das Anregungslicht wird durch das untere Ende der Kapillare in das Reaktionsgefäß eingestrahlt. Ein großer Anteil des emittierten Lichts wird an der Kapillarenwandung reflektiert und gelangt in entgegengesetzter Richtung wieder durch das untere Kapillarenende zur Detektion auf die Photohybride.A system that has been established for several years is the LightCycler instrument (Roche Applied Science Cat. No. 03 531 414 201). The LighCycler device is a Real Time Thermocycler instrument that uses a stream of air to heat or cool capillary reaction vessels. The excitation of fluorescence-labeled hybridization probes or intercalating dyes is carried out with the aid of a light-emitting diode (LED), the detection of fluorescence by means of a fluorimeter containing multiple photo-hybrids for the detection of different wavelengths (WO 97/46712). In this case, both the excitation and the measurement of the emitted light take place by means of a beam path parallel to the longitudinal axis of the capillary (FIG. 1). The excitation light is irradiated through the lower end of the capillary into the reaction vessel. A large proportion of the emitted light is reflected at the Kapillarenwandung and passes in the opposite direction again through the lower capillary end for detection on the photo-hybrid.

Wegen des geringen Durchmessers der Kapillaren sowie des begenzten Detektionswinkels des Fluorimeters muß die Messposition für jede Kapillare exakt eingestellt werden. Die Kapillaren befinden sich in einem drehbaren Karussel als Reaktionsgefäßhalter, der zur Aufnahme von 32 Kapillaren geeignet ist. Jede Kapillare kann mithilfe eines ersten, das Karussel rotierend bewegenden Stepper Motors auf eine bestimmte Messposition in tangentialer Richtung positioniert werden. Ein zweiter Stepper Motor kann das Fluorimeter in eine bestimmte Position gegenüber der jeweils zu messenden Kapillare in radialer Richtung positionieren.Because of the small diameter of the capillaries and the graded detection angle of the fluorimeter, the measurement position for each capillary must be set exactly. The capillaries are in a rotatable carousel as a reaction vessel holder, which is suitable for receiving 32 capillaries. each The capillary can be positioned in a tangential direction using a first stepper motor rotating the carousel. A second stepper motor can position the fluorimeter in a specific position relative to the respective capillary to be measured in the radial direction.

LightCycler Instrumente aller bisherigen Geräteversionen ermitteln zu Beginn eines Experiments für jede einzelne Kapillare die optimale Position für die Fluoreszenzmessung ("Seek"). Dazu wird die mit Reaktionsmix befüllte Kapillare in der Kapillarenhalterung, d.h. bspw im LightCycler Karussel über das Messfenster des Fluorimeters gefahren. Im Bereich der erwarteten Position wird in kleinen Schritten weitergefahren und die Stelle maximaler Fluoreszenz ermittelt. An dieser Stelle wird rechtwinklig dazu das Fluorimeter mit einem zweiten Stepper Motor verschoben und ebenfalls die Position maximaler Fluoreszenz ermittelt. Diese x,y-Position wird für jede Kapillare gespeichert und bei jeder nachfolgenden Messung wieder angefahren.LightCycler instruments of all previous instrument versions determine the optimal position for the fluorescence measurement ("Seek") for each individual capillary at the beginning of an experiment. To do this, the reaction mix filled capillary in the capillary fixture, i. For example, in the LightCycler carousel, drive over the measuring window of the fluorimeter. In the area of the expected position, the procedure continues in small steps and the point of maximum fluorescence is determined. At this point, the fluorimeter is displaced at right angles with a second stepper motor and the position of maximum fluorescence is also determined. This x, y position is stored for each capillary and restarted at each subsequent measurement.

Die als Reaktionsgefäße verwendeten Kapillaren sind so dünn, dass eine hohe Signalintensität nur in einem sehr begrenzten Bereich gemessen wird. Aufgrund von Varianzen in der Kapillarform und der Karussels muss bei jedem Experiment zuerst ein solcher Seek durchgeführt werden (LightCycler Operator's Manual, Version 3.5, Okt. 2000)The capillaries used as reaction vessels are so thin that a high signal intensity is measured only in a very limited range. Because of variances in the capillary shape and the carousels, such a seek must be performed first for each experiment (LightCycler Operator's Manual, Version 3.5, Oct. 2000).

Für den Seek muss das Reaktionsgemisch schon vor der PCR eine ausreichend hohe Grundfluoreszenz aufweisen, sonst kann kein Maximum des Fluoreszenzsignals bestimmt werden. Speziell mit nicht-fluoreszierenden Quenchern, wie sie bei TaqMan-Sonden zunehmend verwendet werden, kann die Grundfluoreszenz so niedrig sein, dass Kapillaren im LightCycler Instrument nicht positioniert werden können.For the Seek, the reaction mixture must have a sufficiently high background fluorescence even before the PCR, otherwise no maximum of the fluorescence signal can be determined. Especially with non-fluorescent quenchers, which are increasingly used in TaqMan probes, the basic fluorescence can be so low that capillaries can not be positioned in the LightCycler instrument.

Aufgabe der vorliegenden Erfindung war es somit, eine Lösung dafür zu etablieren, wie Kapillaren enthaltend eine Reaktionsmischung, die vor Beginn der Reaktion keine oder nur eine zu geringe Fluoreszenz aufweisen, richtig positioniert werden können.It was therefore an object of the present invention to establish a solution for capillaries containing a reaction mixture which have no or only too low fluorescence before the beginning of the reaction can be correctly positioned.

Kurzbeschreibung der ErfindungBrief description of the invention

Diese Aufgabe wird erfindungsgemäß gelöst durch ein Verfahren zur Messung einer Nukleinsäureamplifikation in Real Time, umfassend

  • a) Bereitstellung eines Real Time PCR Instruments, enthaltend
    • einen Reaktionsgefäßhalter zur Aufnahme von multiplen Reaktionsgefäßen
    • Mittel zur Positionierung der multiplen Reaktionsgefäße in einem Reaktionsgefäßhalter relativ zur Detektionseinheit
  • b) Befüllung multipler Reaktionsgefäße mit einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer, mindestens zwei Amplifikations-Primer, mindestens eine Fluoreszenz-markierte Hybridisierungssonde und einer freien Fluoreszenzfarbstoffkomponente mit konstanter Fluoreszenzintensität, die unabhängig von der Amplifikations-Reaktion ist.
  • c) Bestimmung der Position der maximalen Fluoreszenzemission der Fluoreszenzfarbstoffkomponente in multiplen Reaktionsgefäßen in Abhängigkeit von ihrer relativen Position zur Detektionseinheit
  • d) Durchführung der Amplifikationsreaktion und Messung der Fluoreszenzemission der mindestens einen Hybridisationssonde während und optional nach der Amplifikationsreaktion für jedes Reaktionsgefäß an der Position mit maximaler Fluoreszenzemission aus Schritt c)
This object is achieved according to the invention by a method for measuring a nucleic acid amplification in real time, comprising
  • a) providing a Real Time PCR instrument containing
    • a reaction vessel holder for receiving multiple reaction vessels
    • Means for positioning the multiple reaction vessels in a reaction vessel holder relative to the detection unit
  • b) Filling multiple reaction vessels with a reaction mixture containing thermostable polymerase, deoxynucleotides and buffer, at least two amplification primers, at least one fluorescence-labeled hybridization probe and a fluorescence intensity free fluorescent dye component independent of the amplification reaction.
  • c) Determining the position of the maximum fluorescence emission of the fluorescent dye component in multiple reaction vessels as a function of their relative position to the detection unit
  • d) carrying out the amplification reaction and measuring the fluorescence emission of the at least one hybridization probe during and optionally after the amplification reaction for each reaction vessel at the position with maximum fluorescence emission from step c)

Die relative Position des einzelnen Reaktionsgefäßes zur Detektionseinheit kann dadurch verändert werden, daß die Position des Reaktionsgefäßhalters verändert wird. Die relative Position aber auch dadurch verändert werden, dass die Position der Detektionseinheit verändert wird. Darüber hinaus können für eine exakte Positionierung auch beide Positionen verändert werden.The relative position of the individual reaction vessel to the detection unit can be changed by changing the position of the reaction vessel holder. However, the relative position can also be changed by changing the position of the detection unit. In addition, both positions can be changed for exact positioning.

Von der Verwendung eines Referenzfarbstoffs in der PCR-Reaktionsmischung, wie bei ABI-Produkten verwendet (US 5,736,333, Apr. 7, 1998), ist diese Methode klar abgegrenzt. Bei ABI wird der Referenzfarbstoff (ROX) zur Normalisierung der Signalhöhen verschiedener Reaktionsgefäße verwendet, um Varianzen der optischen Messeinheit, der Mikrotiterplatten, sowie der Zusammensetzung und der Füllhöhen der Reagenzienmixe zu kompensierenFrom the use of a reference dye in the PCR reaction mixture as used in ABI products (US 5,736,333, Apr. 7, 1998), this method is clearly defined. ABI uses the reference dye (ROX) to normalize the signal levels of various tubes to compensate for variances in the optical measurement unit, microtiter plates, and composition and fill levels of the reagent mixes

Gegenstand der vorliegenden Erfindung ist auch ein System, bestehend aus

  • einem Real Time PCR Instrument enthaltend Mittel zur Positionierung von multiplen Reaktionsgefäßen in einem Reaktionsgefäßhalter relativ zu einer Detektionseinheit
  • einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer und eine freie Fluoreszenzfarbstoffkomponente, die keine Wechselwirkungen mit der Amplifikationsreaktion eingeht
The subject of the present invention is also a system consisting of
  • a Real Time PCR instrument containing means for positioning multiple reaction vessels in a reaction vessel holder relative to a detection unit
  • a reaction mixture containing thermostable polymerase, deoxynucleotides, buffer and a free fluorescent dye component which does not interact with the amplification reaction

Derartige System enthalten vorzugsweise Kapillaren als Reaktionsgefäße.Such systems preferably contain capillaries as reaction vessels.

Gegenstand der vorliegenden Erfindung ist darüber hinaus eine Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer und zusätzlich einer von der Amplifikationsreaktion unabhängigen Fluoreszenzfarbstoffkomponente mit einer Emissionswellenlänge von mindestens 600 nm.The present invention furthermore relates to a reaction mixture comprising thermostable polymerase, deoxynucleotides, buffers and additionally a fluorescent dye component independent of the amplification reaction and having an emission wavelength of at least 600 nm.

Darüber hinaus enthalten derartige Mischungen vorzugsweise mindestens zwei Amplifikations-Primer und mindestens eine Fluoreszenz-markierte Hybridisierungssonde, beispielsweise eine TaqMan Sonde.In addition, such mixtures preferably contain at least two amplification primers and at least one fluorescence-labeled hybridization probe, for example a TaqMan probe.

Gegenstand der vorliegenden Erfindung ist schließlich auch ein Kit, enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer sowie zusätzlich einer von der Amplifikationsreaktion unabhängigen Fluoreszenzfarbstoffkomponente mit einer Emissionswellenlänger von mindestens 600 nm. Dearartige Kits können zusätzlich spezifische Hybridsierungssonden wie beispielsweise TaqMan Sonden oder auch Amplifikations-Primer enthalten.Finally, the present invention also relates to a kit comprising thermostable polymerase, deoxynucleotides and buffers and additionally to a fluorescence dye component independent of the amplification reaction having an emission wavelength of at least 600 nm. Such kits may additionally contain specific hybridization probes such as TaqMan probes or also amplification primers.

Detaillierte Beschreibung der ErfindungDetailed description of the invention

Die vorliegende Erfindung betrifft in einem ersten Aspekt ein Verfahren zur Messung einer Nukleinsäureamplifikation in Real Time, umfassend

  • a) die Bereitstellung eines Real Time PCR Instruments, enthaltend
    • einen Reaktionsgefäßhalter zur Aufnahme von multiplen Reaktionsgefäßen, und
    • Mittel zur Positionierung der multiplen Reaktionsgefäße in einem Reaktionsgefäßhalter relativ zur Detektionseinheit
  • b) die Befüllung multipler Reaktionsgefäße mit einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer, mindestens zwei Amplifikations-Primer, mindestens eine Fluoreszenz-markierte Hybridisierungssonde und einer Fluoreszenzfarbstoffkomponente, die während und nach der gesamten Amplifikations-Reaktion in freier Form vorliegt,
  • c) die Bestimmung der Position der maximalen Fluoreszenzemission der Fluoreszenzfarbstoff-Komponente in multiplen Reaktionsgefäßen in Abhängigkeit von ihrer relativen Position zur Detektionseinheit, und
  • d) die Durchführung der Amplifikationsreaktion und Messung der Fluoreszenzemission der mindestens einen Hybridisationssonde während und optional nach der Amplifikationsreaktion für jedes Reaktionsgefäß an der Position mit maximaler Fluoreszenzemission aus Schritt c).
The present invention relates in a first aspect to a method for measuring a nucleic acid amplification in real time, comprising
  • a) the provision of a Real Time PCR instrument containing
    • a reaction vessel holder for holding multiple reaction vessels, and
    • Means for positioning the multiple reaction vessels in a reaction vessel holder relative to the detection unit
  • b) the filling of multiple reaction vessels with a reaction mixture containing thermostable polymerase, deoxynucleotides and buffer, at least two amplification primers, at least one fluorescence-labeled hybridization probe and a fluorescent dye component which is in free form during and after the entire amplification reaction,
  • c) the determination of the position of the maximum fluorescence emission of the fluorescent dye component in multiple reaction vessels as a function of their relative position to the detection unit, and
  • d) performing the amplification reaction and measuring fluorescence emission of the at least one hybridization probe during and optionally after the amplification reaction for each reaction tube at the maximum fluorescence emission position of step c).

Der Begriff "Fluoreszenzfarbstoffkomponente" umfasst zunächst jegliche Art von freiem, ungebundenen Fluoreszenzfarbstoff, wobei sich langwellige Fluoreszenzfarbstoffe mit einem Emissionsmaximum von mehr als 600 nm als besonders vorteilhaft erwiesen haben, da die Messung in den kurzwelligeren Detektionskanälen nicht beeinflusst wird. Darüber hinaus umfasst der Begriff "Fluoreszenzfarbstoffkomponente" auch Fluoreszenzfarbstoffe, die an eine weitere Chemische Komponente gekoppelt sind, sofern derartige Moleküle nicht mit der Amplifikationsareaktion interferieren und nicht während der Reaktion in das Amplifikat eingebaut werden.The term "fluorescent dye component" initially includes any type of free, unbound fluorescent dye, with long-wave fluorescent dyes having an emission maximum of more than 600 nm have proven to be particularly advantageous because the measurement is not affected in the shorter wavelength detection channels. In addition, the term "fluorescent dye component" also includes fluorescent dyes which are coupled to another chemical component, if such molecules do not interfere with the Amplification reaction interfere and not be incorporated during the reaction in the amplicon.

Aufgrund der Tatsache, dass sich beispielsweise beim LightCycler Karussel nicht nur die einzelnen Bohrungen für die Kapillaren, sondern auch die Kapillaren untereinander in ihren Dimensionen zumindest innerhalb gewisser Grenzen unterscheiden, ist eine Positionierung für jede einzelnen Kapillare vor jedem Experiment notwendig. Essentiell ist ein derartiges Verfahren, wenn als Reaktionsgefäße Kapillaren verwendet werden, da ohne eine exakte Positionierung derartiger Reaktionsgefäße im Strahlengang der Detektionseinheit, d. h. des Fluorimeters, keine zuverlässige Fluoreszenzmessung durchgeführt werden kann.Due to the fact that, for example, in the LightCycler carousel not only the individual bores for the capillaries, but also the capillaries differ in their dimensions at least within certain limits, a positioning for each capillary before each experiment is necessary. Such a method is essential if capillaries are used as reaction vessels, since without an exact positioning of such reaction vessels in the beam path of the detection unit, ie. H. of the fluorimeter, no reliable fluorescence measurement can be performed.

Bei dem einen Mittel oder mehreren Mitteln zur Positionierung multipler Reaktionsgefäße in einem Reaktionsgefäßhalter relativ zur Detektionseinheit kann es sich erfindungsgemäß um jegliche Art von fein justierbaren Antrieb handeln. Vorzugsweise handelt es sich dabei um einen oder mehre Stepper Motoren, die in sehr kleinen Schritten entweder die Position des Reaktionsgefäßhalters oder die Position der Detektionseinheit oder beide verändern können.The one or more means for positioning multiple reaction vessels in a reaction vessel holder relative to the detection unit may according to the invention be any type of finely adjustable drive. Preferably, these are one or more stepper motors, which can change in very small steps either the position of the reaction vessel holder or the position of the detection unit or both.

Erfindungsgemäß kann es sich bei der Detektionseinheit um jegliche Art von Detektoren handeln, die in der Lage sind, Fluoreszenzsignale zu detektieren. Dazu gehören beispielsweise Fluorimeter mit einem oder mehreren Photohybrid Dioden oder auch CCD Kameras.According to the invention, the detection unit may be any type of detectors capable of detecting fluorescence signals. These include, for example, fluorimeter with one or more photo-hybrid diodes or CCD cameras.

Detektionsformatedetection formats

Das oben beschriebene LightCycler Instrument kann für verschiedene Detektionsformate benutzt werden:The LightCycler instrument described above can be used for various detection formats:

a) Interkalierender DNA-Bindefarbstoff:a) Intercalating DNA binding dye:

Das jeweilige Amplifikationsprodukt wird in diesem Fall durch einen DNA-Bindefarbstoff nachgewiesen, welcher bei Interaktion mit doppelsträngiger Nukleinsäure nach Anregung mit Licht einer geeigneten Wellenlänge ein entsprechendes Fluoreszenzsignal emitiert. Als besonders geeignet für diese Anwendung haben sich die Farbstoffe SybrGreen und SybrGold (Molecular Probes) erwiesen. Die vorhandene Grundfluoreszenz des SybrGreen vor Beginn einer Amplifikationsreaktion reicht in der Regel aus, um einen Positionierungsprozess durchzuführen.The respective amplification product is detected in this case by a DNA binding dye, which emits a corresponding fluorescence signal upon interaction with double-stranded nucleic acid after excitation with light of a suitable wavelength. Particularly suitable for this application are the dyes SybrGreen and SybrGold (Molecular Probes) proved. The existing basic fluorescence of SybrGreen prior to the start of an amplification reaction is usually sufficient to perform a positioning process.

b) FRET-Hybridisierungssonden:b) FRET hybridization probes:

Für dieses Testformat werden zwei einzelsträngige Hybridisierungssonden gleichzeitig verwendet, die komplementär zu benachbarten Stellen desselben Strangs der amplifizierten Target-Nukleinsäure sind. Beide Sonden sind mit unterschiedlichen Fluorophoren markiert, die sich bezüglich ihrer Extinktions- und Emissionswellenlängen unterscheiden. Bei Anregung des kurzwelligeren Fluorophors ist ohne Target-Nukleinsäure fast ausschließlich die Fluoreszenz dieses Fluorophors messbar. Hybridisieren beide Sonden an amplifizierte Target-Nukleinsäure, nähern sich die beiden Fluorophore soweit an, daß der angeregte Fluorophor nach dem Prinzip des Fluoreszenzresonanzenergietransfers die absorbierte Energie auf den zweiten Fluorophor übertragen kann und dessen Emission im langwelligen Detektionskanal messbar wird (WO 97/46707). Dabei wird als sogenannte Donor-Komponente beispielsweise Fluoreszein, mit Licht einer geeigneten Wellenlänge angeregt. Bei räumlicher Nähe zu einer geeigneten Akzeptorkomponente wie beispielsweise bestimmten Rhodamin-Derivaten erfolgt dann ein Resonanzenergietransfer auf die Akzeptorkomponente, so daß der Akzeptorfluorophor Licht einer höheren Emissionswellenlänge emittiert. Auch für dieses Format ist die Emission des FRET Donors hinreichend, um einen Positionierungsprozess durchführen zu können.For this assay format, two single-stranded hybridization probes are used simultaneously which are complementary to adjacent sites of the same strand of amplified target nucleic acid. Both probes are labeled with different fluorophores, which differ in their absorbance and emission wavelengths. When the shorter-wavelength fluorophore is excited, almost exclusively the fluorescence of this fluorophore can be measured without target nucleic acid. If both probes hybridize to amplified target nucleic acid, the two fluorophores approach so far that the excited fluorophore can transfer the absorbed energy to the second fluorophore according to the principle of fluorescence resonance energy transfer and its emission in the long-wave detection channel becomes measurable (WO 97/46707). In this case, as a so-called donor component, for example, fluorescein is excited with light of a suitable wavelength. In close proximity to a suitable acceptor component, such as certain rhodamine derivatives, resonance energy transfer then takes place to the acceptor component, so that the acceptor fluorophore emits light of a higher emission wavelength. Also for this format, the emission of the FRET donor is sufficient to perform a positioning process.

c) TaqMan-Hybridisierungssondenc) TaqMan hybridization probes

Eine einzelsträngige Hybridisierungssonde wird mit 2 Komponenten markiert. Bei Anregung der ersten Komponente mit Licht einer geeigneten Wellenlänge wird nach dem Prinzip des Fluoreszenzresonanzenergietransfers die absorbierte Energie auf die zweite Komponente, den sogenannten Quencher übertragen, so daß die Strahlungsemission der ersten Komponente fast vollständig unterdrückt wird. Während des Annealing Schrittes der PCR Reaktion bindet die Hybridisationssonde an die Target-DNA und wird bei der Primerelongation durch die 5'-3' Exonuklease-Aktivität der Taq-Polymerase gespalten. Dadurch werden die angeregte Fluoreszenzkomponente und der Quencher räumlich voneinander getrennt, so dass eine Fluoreszenzemission der ersten Komponente bei der entsprechenden Wellenlänge gemessen werden kann (US 5,210,015, US 5,487,972, US 5,804,375, US 6,214,979).A single-stranded hybridization probe is labeled with 2 components. Upon excitation of the first component with light of a suitable wavelength, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer, so that the radiation emission of the first component is almost completely suppressed. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is cleaved upon primer elongation by the 5'-3 'exonuclease activity of the Taq polymerase. This will be the excited fluorescent component and the quencher are spatially separated so that a fluorescence emission of the first component at the corresponding wavelength can be measured (US 5,210,015, US 5,487,972, US 5,804,375, US 6,214,979).

Aufgrund der Tatsache, daß derartige Hybridisierungssonden mit nicht-fluoreszierenden Quenchern in ungebundenem, intakten Zustand selbst bei Anregung der ersten Komponente praktisch keine Fluoreszenzemission ausstrahlen, ist es für dieses Format essentiell, die Reaktionsmischung erfindungsgemäß mit einer zusätzlichen, schon vor der Amplifikation detektierbaren Farbstoffkomponente zu versehen, mit deren Hilfe eine Positionierung der Reaktionsgefäße im LightCycler erfolgen kann.Due to the fact that such hybridization probes emit virtually no fluorescence emission with non-fluorescent quenchers in the unbound, intact state even when the first component is excited, it is essential for this format to provide the reaction mixture with an additional dye component detectable even before the amplification , which can be used to position the reaction vessels in the LightCycler.

Zusatz einer Fluoreszenzfarbstoff-KomponenteAddition of a fluorescent dye component

Für eine sichere Positionierung der LightCycler Kapillaren, besonders bei Reaktionsgemischen mit geringer Grundfluoreszenz wie den TaqMan-Sonden mit nicht fluoreszierenden Quenchern, wird deshalb erfindungsgemäß der Reaktionsmischung ein passiver Fluoreszenzfarbstoff ("Seek Dye") zugesetzt, der die PCR nicht beeinflusst und die optische Detektion nicht stört. Mit anderen Worten, die Reaktionsmischung enthält erfindungsgemäß eine Fluoreszenzfarbstoffkomponente, die während und nach der gesamten Amplifikationsreaktion in freier Form vorliegt. Das bedeutet, dass die Farbstoffkomponente nicht in Form von markierten Primern oder markierten dNTPs während der Amplifikation kovalent in die Amplifikationsprodukte eingebaut wird und darüber hinaus auch keine Bindungsaffinität zum doppelsträngigen DNA Amplifikationsprodukt aufweist, wie das bspw. bei SybrGreen der Fall ist.For a secure positioning of the LightCycler capillaries, especially in reactions with low background fluorescence such as the TaqMan probes with non-fluorescent quenchers, according to the invention a passive fluorescence dye ("Seek Dye") is added to the reaction mixture, which does not affect the PCR and optical detection is not disturbs. In other words, according to the invention, the reaction mixture contains a fluorescent dye component which is in free form during and after the entire amplification reaction. This means that the dye component is not covalently incorporated into the amplification products in the form of labeled primers or labeled dNTPs during amplification and moreover also has no binding affinity for the double-stranded DNA amplification product, as is the case with SybrGreen, for example.

Das Emissionsmaximum dieses passiven Seek Dye liegt vorzugsweise im langwelligen Bereich von mehr als 600 nm und besonders bevorzugt im langwelligsten Detektionskanal des sechskanäligen LightCycler 2.0 Instruments. Die Detektion in den kurzwelligeren Kanälen 530 - 640 nm, die bei LightCycler TaqMan Assays aufgrund der kommerziell verfügbaren und geeigneten Farbstoffe fast ausschließlich verwendet werden, wird dadurch nicht beeinflusst. In den Kanälen 705 und 670 nm ist lediglich eine konstante Erhöhung des Hintergrundsignals sichtbar, die bei der Messung von Signalhöhenveränderungen in der Regel nicht stört. Der Seek Dye besitzt eine ausreichend hohe Stabilität gegen Temperatur- und Lichtbelastung, so dass sich die Eigenschaften während der PCR nicht verändern. Besonders bevorzugt hat sich in diesem Zusammenhang die Verwendung von JA286 (EP 0 747 447, example 1) erwiesen.The emission maximum of this passive Seek Dye is preferably in the long-wavelength range of more than 600 nm and particularly preferably in the longest-wave detection channel of the six-channel LightCycler 2.0 instrument. The detection in the shorter wave channels 530-640 nm, in LightCycler TaqMan assays due to the commercially available and suitable dyes almost exclusively used will not be affected. In the channels 705 and 670 nm only a constant increase of the background signal is visible, which usually does not interfere with the measurement of signal level changes. The Seek Dye has a sufficiently high stability against temperature and light exposure, so that the properties do not change during the PCR. In this context, the use of JA286 (EP 0 747 447, example 1) has proven to be particularly preferred.

Gegenstand der vorliegenden Erfindung ist auch ein System, bestehend aus

  • einem Real Time PCR Instrument enthaltend Mittel zur Positionierung von multiplen Reaktionsgefäßen in einem Reaktionsgefäßhalter relativ zu einer Detektionseinheit
  • einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer und eine Fluoreszenzfarbstoff-Komponente, wobei die Fluoreszenzfarbstoffkomponente während und nach einer gesamten Amplifikationsreaktion in vollständig freier Form vorliegt.
The subject of the present invention is also a system consisting of
  • a Real Time PCR instrument containing means for positioning multiple reaction vessels in a reaction vessel holder relative to a detection unit
  • a reaction mixture comprising thermostable polymerase, deoxynucleotides, buffers and a fluorescent dye component, wherein the fluorescent dye component is in completely free form during and after an entire amplification reaction.

Bevorzugt enthält das erfindungsgemäße System Kapillaren als Reaktionsgefäße.The system according to the invention preferably contains capillaries as reaction vessels.

Ebenfalls bevorzugt enthält die Reaktionsmischung bereits mindestens zwei Amplifikationsprimer sowie mindestens eine Hybridisierungssonde, beispielsweise eine TaqMan- Hybridisierungssonde.Also preferably, the reaction mixture already contains at least two amplification primers and at least one hybridization probe, for example a TaqMan hybridization probe.

Darüber hinaus enthält das System als Fluoreszenzfarbstoff-Komponente bevorzugt einen Fluoreszenzfarbstoff mit einem Emissionsmaximum von mehr als 600 nm, beispielsweise den Fluoreszenzfarbstoff JA 286 (EP 0 747 447, example 1).In addition, the system preferably contains as fluorescent dye component a fluorescent dye with an emission maximum of more than 600 nm, for example the fluorescent dye JA 286 (EP 0 747 447, example 1).

Positionierungsprozesspositioning process

Der Positionierungsprozess (Seek Verfahren) im Light Cycler kann für eine einzelne Kapillare wie folgt verlaufen:The positioning process (Seek procedure) in the Light Cycler can be as follows for a single capillary:

Vor jedem Experiment (Run) müssen von allen Kapillaren mit den darin enthaltenen Reaktionsmischungen die optimalen Messpositionen in radialer und tangentialer Richtung bestimmt werden. Diese Positionen gelten immer für einen gesamten Run und müssen bei jedem Neustart eines Protokolls neu bestimmt werden.Before each experiment (run), all capillaries with the reaction mixtures contained therein must determine the optimal measuring positions in the radial and tangential direction. These positions always apply to one entire run and must be redetermined each time a log is restarted.

Die geschlossene Kammer wird auf 30°C oder eine andere im Run vorgegebene Temperatur aufgeheizt. Nach Erreichen der Temperatur wird ein Home-Lauf durchgeführt, bei dem die Lichtschranken für Photometer und Rotor als Referenzposition angefahren werden. Danach wird für jede Probe die Position des maximalen Fluoreszenzsignals bestimmt. Die Messung erfolgt in allen vorhandenen Detektionskanälen, der Kanal mit dem höchsten Signal wird ausgewertetThe closed chamber is heated to 30 ° C or another temperature set in Run. When the temperature has been reached, a home run is performed in which the photoelectric sensors and rotor are approached as the reference position. Thereafter, the position of the maximum fluorescence signal is determined for each sample. The measurement takes place in all existing detection channels, the channel with the highest signal is evaluated

In einem EEPROM genannten Speicherplatz ist die Sollposition für Kapillare 1 als CarOffset und RadOffset gespeichert. Um diese Position wird ein Fenster gespannt, in dem der Seek für Kapillare 1 erfolgt. Die Messung erfolgt entlang 4 kreuzender Bahnen ('#'), daher wird dieser Vorgang als Cross-Seek bezeichnet. Für jede folgende Kapillare wird das Probenkarussel von der jeweils bestimmten Position um 1/32 des Umfangs weitergedreht und erneut der Cross-Seek durchgeführt.In a storage space named EEPROM, the setpoint position for capillary 1 is stored as CarOffset and RadOffset. Around this position, a window is stretched, in which the Seek for capillary 1 takes place. The measurement takes place along 4 crossing tracks ('#'), therefore this process is called cross-seek. For each following capillary, the sample carousel is rotated further from the respectively determined position by 1/32 of the circumference and again the cross-seek is performed.

Der Cross-Seek erfolgt in 4 Phasen:The cross-Seek takes place in 4 phases:

Phase 1 = KarussellPhase 1 = carousel

Durch Drehen des Probenkarussells in kleinen Schritten wird innerhalb des definierten Fensters um die angefahrene Position die Karusselposition des maximalen Signals bestimmt und gespeichert.By turning the sample carousel in small steps, the carousel position of the maximum signal is determined and stored within the defined window around the approached position.

Phase 2 = PhotometerPhase 2 = photometer

Bei der maximalen Rotorposition von Phase 1 wird innerhalb des definierten Fensters die Photometerposition des maximalen Signals bestimmt und gespeichert.At the maximum rotor position of phase 1, the photometer position of the maximum signal is determined and stored within the defined window.

Phase 3 = KarussellPhase 3 = carousel

Bei der maximalen Photometerposition von Phase 2 wird innerhalb des neuen Fensters um die Karusselposition aus Phase 1 erneut die Karusselposition des maximalen Signals bestimmt und gespeichert.At the maximum photometer position of phase 2, within the new window around the carousel position from phase 1, the carousel position of the maximum signal is again determined and stored.

Phase 4 = PhotometerPhase 4 = photometer

Bei der maximalen Karusselposition von Phase 3 wird innerhalb des neuen Fensters um die Photometerposition aus Phase 2 erneut die Photometerposition des maximalen Signals bestimmt und gespeichert.At the maximum carousel position of phase 3, the photometer position of the maximum signal is again determined and stored within the new window around the photometer position from phase 2.

Nur wenn die maximale Fluoreszenz einen vorgegebenen Minimalwert überschreitet, wird diese Kapillare als gefunden deklariert. Ist das Signal zu gering, gilt diese Kapillare als nicht gefunden. Damit wird vermieden, dass Positionen ohne Reaktionsmischung unsinnig positioniert oder fälschlich ausgewertet werden.Gilt die Kapillare als gefunden, werden die ermittelten Koordinaten von Rotor und Photometer der Phase-4 gespeichert und bei der Vermessung der Kapillare im folgenden RUN jeweils angefahren.Only if the maximum fluorescence exceeds a given minimum value, this capillary is declared as found. If the signal is too low, this capillary is considered not found. This avoids that positions without reaction mixture are nonsensitively positioned or incorrectly evaluated. If the capillary is found, the determined coordinates of rotor and photometer of phase-4 are stored and approached each time during the measurement of the capillary in the following RUN.

Mischungen und KitsMixtures and kits

In einem weiteren Aspekt beinhaltet die vorliegende Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer, wobei zusätzlich eine Fluoreszenzfarbstoffkomponente enthalten ist, die während und nach der gesamten Amplifikationsreaktion in freier Form vorliegt. Vorzugsweise handelt es sich dabei um einem Farbstoff mit einer Emissionswellenlänge von mehr als 600 nm. Besonders bevorzugt ist der Fluoreszenzfarbstoff JA286.In a further aspect, the present reaction mixture containing thermostable polymerase, deoxynucleotides, comprises buffers, additionally containing a fluorescent dye component which is in free form during and after the entire amplification reaction. Preferably, this is a dye having an emission wavelength of more than 600 nm. Particularly preferred is the fluorescent dye JA286.

Derartige Mischungen können als Master Mixe für multiple Reaktionen hergestellt werden. Nach entsprechender Aliquotierung können derartige Mischungen vor dem endgültigen Einsatz in einer Amplifikationsreaktion noch mit mindestens 2 spezifischen Amplifikationsprimern sowie mit mindestens einer Fluoreszenzmarkierten Hybridisationssonde wie beispielsweise einer TaqMan-Sonde versehen werden.Such mixtures can be prepared as master mixes for multiple reactions. After appropriate aliquoting such mixtures may be provided before the final use in an amplification reaction with at least 2 specific amplification primers and with at least one fluorescently labeled hybridization probe such as a TaqMan probe.

Bestandteil der vorliegenden Erfindung sind darüber hinaus Kits, enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer sowie zusätzlich eine Fluoreszenzfarbstoffkomponente mit einer Emissionswellenlänge von mindestens 600 nm, die während und nach der gesamten Amplifikationsreaktion in vollständig freier Form vorliegt. Vorzugsweise handelt es sich dabei ebenfalls um einem Farbstoff mit einer Emissionswellenlänge von mehr als 600 nm. Besonders bevorzugt ist der Fluoreszenzfarbstoff JA286.The present invention furthermore relates to kits comprising thermostable polymerase, deoxynucleotides and buffers, and additionally a fluorescent dye component having an emission wavelength of at least 600 nm, which is present in completely free form during and after the entire amplification reaction. Preferably, this is also a Dye with an emission wavelength of more than 600 nm. Particularly preferred is the fluorescent dye JA286.

Dabei sind zwei Arten von Kits zu unterscheiden:There are two types of kits:

Generische Kits enthalten keine weiteren Bestandteile und können somit für jegliche Art von Real Time PCR unabhängig vom verwendeten Detektionsformat und unabhängig von der zu amplifizierenden Target Sequenz verwendet werden. Der Endverbraucher muß also in diesem Falle noch auf individueller Basis Primer und Hybridisierungssonden gemäß seinen Anforderungen hinzufügen.Generic kits contain no additional components and can therefore be used for any type of real-time PCR, regardless of the detection format used and regardless of the target sequence to be amplified. The end user must add in this case still on an individual basis primers and hybridization probes according to his requirements.

Parameterspezifische Kits enthalten zusätzlich in der Regel mindestens zwei Amplifikationsprimer sowie mindestens eine Fluoreszenz-markierte Hybridisierungsssonde, vorzugsweise eine TaqMan Sonde.In addition, parameter-specific kits usually contain at least two amplification primers and at least one fluorescence-labeled hybridization probe, preferably a TaqMan probe.

Einen Sonderfall stellen sogenannte ASRs (Analyte specific reagents) dar. Derartige Kits enthalten mindestens zwei Amplifikationsprimer, mindestens eine Fluoreszenz-markierte Hybridisierungssonde sowie erfindungsgemäß eine Fluoreszenzfarbstoffkomponente.So-called ASRs (analyte-specific reagents) are a special case. Such kits contain at least two amplification primers, at least one fluorescence-labeled hybridization probe and, according to the invention, a fluorescent dye component.

Beschreibung des SequenzprotokollsDescription of the Sequence Listing Beschreibung der FigurenDescription of the figures

Figur 1FIG. 1
Aufbau des LightCycler InstrumentsConstruction of the LightCycler instrument
Figur 2FIG. 2
Amplifiktionskurven einer Cyclophilin A PCR in Gegenwart von unterschiedlichen JA286 Konzentrationen.Amplification curves of a cyclophilin A PCR in the presence of different JA286 concentrations.
Figur 3FIG. 3
Cyclophilin A PCR mit verschiedenen Mengen an humaner genomischer DNA in Gegenwart des Farbstoffs JA286Cyclophilin A PCR with various amounts of human genomic DNA in the presence of the dye JA286

Die folgenden Beispiele, Publikationen, das Sequenzprotokoll und die Abbildungen erläutern die Erfindung, deren Schutzumfang sich aus den Patentansprüchen ergibt, weiter. Die beschriebenen Verfahren sind als Beispiele zu verstehen, die auch noch nach Modifikationen den Gegenstand der Erfindung beschreiben.The following examples, publications, the sequence listing and the figures further illustrate the invention, the scope of which is set forth in the claims. The methods described are to be understood as examples which describe the subject matter of the invention even after modifications.

Beispiel 1example 1

In einem LightCylcer Experiment wurde ausgehend von 300 pg humaner genomischer DNA ein 442 bp Fragment des Cyclophillin A Gens mit folgenden Primern nach einem Standard Thermocycling Protokoll in Gegenwart von verschiedenn Mengan des Fluoreszenzfarbsotoffs JA 286 (EP 0 747 447, example 1) amplifiziert:

  • Primer:
    Figure imgb0001
    Figure imgb0002
In a LightCylcer experiment, starting from 300 pg of human genomic DNA, a 442 bp fragment of the cyclophillin A gene was amplified with the following primers according to a standard thermocycling protocol in the presence of different Mengan of the fluorescence colorant JA 286 (EP 0 747 447, example 1):
  • primer:
    Figure imgb0001
    Figure imgb0002

Dabei wurde unter den folgenden Bedingungen amplifiziert: Program Cycles Analysis Mode Target Temp. [°C] Hold [hh:mm:ss] Trans. Rate [°C/s] Sec. Target Step Size [°C] Step Delay [Cycl.] Acquisition Mode Heating 1 None 95 00:10:00 20 0 0 0 None Cycling 45 Quantif. 95 00:00:10 20 0 0 0 None 55 00:00:30 20 0 0 0 None 72 00:00:01 20 0 0 0 Single Cooling 1 40 00:00:30 20 0 0 0 None It was amplified under the following conditions: Program Cycles Analysis Mode Target Temp. [° C] Hold [hh: mm: ss] Trans. Rate [° C / s] Sec. target Step Size [° C] Step Delay [Cycl.] Acquisition Mode Heating 1 none 95 0:10:00 20 0 0 0 none Cycling 45 Quantif. 95 0:00:10 20 0 0 0 none 55 0:00:30 20 0 0 0 none 72 0:00:01 20 0 0 0 single Cooling 1 40 0:00:30 20 0 0 0 none

Das Amplifikatiosnprodukt wurde mithilfe der folgenden TaqMan Sonde im Real Time Modus detektiert:

Figure imgb0003
The amplified product was detected using the following TaqMan probe in real-time mode:
Figure imgb0003

Bereits bei Zugabe von 0.5 µM JA286 war eine exakte Kapillarenpositionierung erfolgreich.Precise capillary positioning was successful even with the addition of 0.5 μM JA286.

Wie in Fig. 2 dargestellt, konnte auch bei einer Zugabe von bis zu 50 mm µM J 286 keine Inhibition der Amplifikationsreaktion festgestellt werden.As shown in Fig. 2, no inhibition of the amplification reaction could be detected even with an addition of up to 50 mm μM J 286.

Beispiel 2Example 2

In einem weiteren Experiment analog zu Beispiel 1 wurden bei Zugabe von jeweils einer konstanten Menge von 0,5µM JA 286 Farbstoff veschiedene Mengen (3pg, 30 pg, 300pg, 3ng, 30 ng) an genomischer Template DNA zur Amplifikation des Cyclophillin A Gens verwendet. Wie aus Fig. 3 ersichtlich, führt die Anwesenheit des zusätzlichen Farbstoffs weder bei niedrigen noch bei hohen Template Konzentrationen zu einer Verfälschung des quantitativen Ergebnisses.In a further experiment analogous to Example 1, JA 286 dye different amounts (3pg, 30 pg, 300pg, 3ng, 30 ng) of genomic template DNA were used to amplify the cyclophillin A gene with the addition of a constant amount of 0.5 .mu.M. As can be seen from Fig. 3, the presence of the additional dye, either at low or at high template concentrations leads to a falsification of the quantitative result.

ReferenzlisteReferences

  • EP 0 747 447EP 0 747 447
  • LightCycler Operator's Manual, Version 3.5, Okt. 2000LightCycler Operator's Manual, Version 3.5, Oct. 2000
  • US 5,210,015US 5,210,015
  • US 5,487,972US 5,487,972
  • US 5,736,333US 5,736,333
  • US 5,804,375US 5,804,375
  • US 6,214,979US 6,214,979
  • WO 97/46707WO 97/46707
  • WO 97/46712WO 97/46712
Annex zu den Anmeldungsunterlagen - nachgereichtes SequenzprotokollAnnex to the application documents - Subsequent Sequence Listing

Figure imgb0004
Figure imgb0005
Figure imgb0004
Figure imgb0005

Claims (10)

Verfahren zur Messung einer Nukleinsäureamplifikation in Real Time, umfassend a) Bereitstellung eines Real Time PCR Instruments, enthaltend - einen Reaktionsgefäßhalter zur Aufnahme von multiplen Reaktionsgefäßen - Mittel zur Positionierung der multiplen Reaktionsgefäße in einem Reaktionsgefäßhalter relativ zur Detektionseinheit b) Befüllung multipler Reaktionsgefäße mit einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer, mindestens zwei Amplifikations-Primer, mindestens eine Fluoreszenz-markierte Hybridisationssonde und einer Fluoreszenzfarbstoffkomponente, die während und nach der gesamten Amplifikations-Reaktion in freier Form vorliegt c) Bestimmung der Position der maximalen Fluoreszenzemission der Fluoreszenzfarbstoffkomponente in multiplen Reaktionsgefäßen in Abhängigkeit von ihrer relativen Position zur Detektionseinheit d) Durchführung der Amplifikationsreaktion und Messung der Fluoreszenzemission der mindestens einen Hybridisationssonde während und optional nach der Amplifikationsreaktion für jedes Reaktionsgefäß an der Position mit maximaler Fluoreszenzemission aus Schritt c) A method for measuring a nucleic acid amplification in real time, comprising a) providing a Real Time PCR instrument containing - A reaction vessel holder for receiving multiple reaction vessels - Means for positioning the multiple reaction vessels in a reaction vessel holder relative to the detection unit b) Filling multiple reaction vessels with a reaction mixture containing thermostable polymerase, deoxynucleotides and buffer, at least two amplification primers, at least one fluorescence-labeled hybridization probe and a fluorescent dye component which is in free form during and after the entire amplification reaction c) Determining the position of the maximum fluorescence emission of the fluorescent dye component in multiple reaction vessels as a function of their relative position to the detection unit d) carrying out the amplification reaction and measuring the fluorescence emission of the at least one hybridization probe during and optionally after the amplification reaction for each reaction vessel at the position with maximum fluorescence emission from step c) Verfahren nach Anspruch 1 dadurch gekennzeichnet, dass die Position des Reaktionsgefäßhalters verändert wirdA method according to claim 1, characterized in that the position of the reaction vessel holder is changed Verfahren nach Anspruch 1 dadurch gekennzeichnet, dass die Position der Detektionseinheit verändert wirdA method according to claim 1, characterized in that the position of the detection unit is changed System, bestehend aus - einem Real Time PCR Instrument enthaltend Mittel zur Positionierung von multiplen Reaktionsgefäßen in einem Reaktionsgefäßhalter relativ zu einer Detektionseinheit - einer Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer und eine Fluoreszenzfarbstoffkomponente
dadurch gekennzeichnet, daß
die Fluoreszenzfarbstoffkomponente während und nach einer gesamten Amplifikationsreaktion in vollständig freier Form vorliegtSystem consisting of - A Real Time PCR instrument containing means for positioning of multiple reaction vessels in a reaction vessel holder relative to a detection unit a reaction mixture containing thermostable polymerase, deoxynucleotides, buffer and a fluorescent dye component
characterized in that
the fluorescent dye component is in completely free form during and after an entire amplification reaction System nach Anspruch 4, dadurch gekennzeichnet, daß die Reaktionsgefäße Kapillaren sindSystem according to claim 4, characterized in that the reaction vessels are capillaries Reaktionsmischung enthaltend thermostabile Polymerase, Deoxynukleotide, Puffer,
dadurch gekennzeichnet, daß
zusätzlich eine Fluoreszenzfarbstoffkomponente mit einer Emissionswellenlänger von mindestens 600 nm enthalten ist, die während und nach der gesamten Amplifikationsreaktion in freier Form vorliegtReaction mixture containing thermostable polymerase, deoxynucleotides, buffers,
characterized in that
additionally a fluorescent dye component with an emission wavelength of at least 600 nm is present, which is in free form during and after the entire amplification reaction Reaktionsmischung nach Anspruch 6, zusätzlich enthaltend mindestens zwei Amplifikations-Primer und mindestens eine Fluoreszenz-markierte HybridisationssondeReaction mixture according to claim 6, additionally containing at least two amplification primers and at least one fluorescence-labeled hybridization probe Reaktionsmischung nach Anspruch 7, enthaltend mindestens eine TaqManSondeReaction mixture according to claim 7, containing at least one TaqMan probe Kit, enthaltend thermostabile Polymerase, Deoxynukleotide und Puffer
dadurch gekennzeichnet, daß
zusätzlich eine Fluoreszenzfarbstoffkomponente mit einer Emissionswellenlänge von mindestens 600 nm enthalten ist, die während und nach der gesamten Amplifikationsreaktion in vollständig freier Form vorliegtKit containing thermostable polymerase, deoxynucleotides and buffers
characterized in that
additionally a fluorescent dye component with an emission wavelength of at least 600 nm is present, which is in completely free form during and after the entire amplification reaction Kit nach Anspruch 9, zusätzlich enthaltend mindestens eine Fluoreszenz-markierte Hybridisationssonde, vorzugsweise eine TaqMan SondeKit according to claim 9, additionally containing at least one fluorescence-labeled hybridization probe, preferably a TaqMan probe
EP04019542A 2004-08-18 2004-08-18 Method for detecting nucleic acid amplification in real-time comprising positioning a reaction vial relative to a detection unit Not-in-force EP1627921B1 (en)

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